The overall goal of this project is to develop novel biomarkers and diagnostic strategies for the early detection of occult urinary bladder neoplasia and its progression from intraurothelial preneoplastic conditions to invasive cancer. The current proposal represents an extension and logical continuation of the studies performed during the first cycle of the Early Detection Research Network (EDRN). These studies have led to the identification of six chromosomal regions critical for the development of bladder cancer and have provided evidence for the existence of a novel class of genes referred to as "forerunners" whose involvement precedes the loss of function of major tumor suppressor genes in the development of cancer. We hypothesize that in situ clonal expansion of bladder preneoplasia is caused by loss of function of the forerunner genes mapping contiguously to known major tumor suppressors such as RB1. Our in vitro studies utilizing short interference RNA provide evidence that loss of function of these genes is critical for growth advantage of urothelial cells. Thus, the identification of novel forerunner genes and the investigation of their involvement in early occult phases of human bladder preneoplasia may not only provide important mechanistic clues to the development of human cancer but may also identify a novel class of early detection markers capable of detecting the clinically and microscopically occult phases of human cancer development. We also hypothesized a complementary mechanism of bladder cancer progression based on the amplification and overexpression of a recently identified oncogenic kinase STK15/BTAK/Aurora-A shown to be involved in regulating chromosomal segregation and centrosome function and more recently to be capable of destabilizing the p53 tumor suppressor protein. In this grant we propose the following major groups of studies: (1) Perform functional analyses and assessment of the already identified putative "forerunner genes" (ITM2B, P2RY5, and CHClL) located contiguously to RB1 as novel early detection biomarkers. Additionally, we propose to characterize other predicted forerunner gene harboring loci mapping on chromosomes 17p13 and 5q22-23 and to assess the candidate genes identified as biomarkers for bladder cancer detection. We will also investigate the contribution of genetic and epigenetic mechanisms in the functional inactivation of each of the genes to elucidate a comprehensive view of the role of forerunner genes in the malignant transformation process that will enhance our ability to detect forerunner genes and provide clinically relevant markers for early detection. (2) Extension of our studies on STK15/BTAK/Aurora-A and its interacting genes involved in the pathway as biomarkers for bladder cancer detection. (3) Combine the STK15/BTAK/Aurora-A and its regulatory partners with the forerunner genes in a panel of approximately 15 markers testing their ability to identify occult bladder neoplasia in a prospective validation trial. Since both major categories of novel genes, i.e. the forerunner genes and Aurora-A with its partners, appear to be universally involved in human carcinogenesis the completion of this project will have major relevance for early detection of other common human cancers.